Flow injection combined with ICP-MS for accurate high throughput analysis of elemental impurities in pharmaceutical products according to USP <232>/<233>.

New guidelines of the United States Pharmacopeia (USP), European Pharmacopeia (EP) and international organization (ICH, International Conference on Harmonization) regulating elemental impurity limits in pharmaceuticals seal the end of unspecific analysis of metal(oid)s as outlined in USP <231> and EP 2.4.8. Chapter USP <232> and EP 5.20 as well as drafts from ICH Q3D specify both daily doses and concentration limits of metallic impurities in pharmaceutical final products and in active pharmaceutical ingredients (API) and excipients. In chapters USP <233> and EP 2.4.20 method implementation, validation and quality control during the analytical process are described. By contrast with the--by now--applied methods, substance specific quantitative analysis features new basic requirements, further, significantly lower detection limits ask for the necessity of a general changeover of the methodology toward sensitive multi element analysis by ICP-AES and ICP-MS, respectively. A novel methodological approach based on flow injection analysis and ICP-SFMS/ICP-QMS for the quick and accurate analysis of Cd, Pb, As, Hg, Ir, Os, Pd, Pt, Rh, Ru, Cr, Mo, Ni, V, Cu, Mn, Fe and Zn in drug products by prior dilution, dissolution or microwave assisted closed vessel digestion according to the regulations is presented. In comparison to the acquisition of continuous signals, this method is advantageous with respect to the unprecedented high sample throughput due to a total analysis time of approximately 30s and the low sample consumption of below 50 µL, while meeting the strict USP demands on detection/quantification limits, precision and accuracy.

Dominant-negative inhibition of the Axl receptor tyrosine kinase suppresses brain tumor cell growth and invasion and prolongs survival.

Malignant gliomas remain incurable brain tumors because of their diffuse-invasive growth. So far, the genetic and molecular events underlying gliomagenesis are poorly understood. In this study, we have identified the receptor tyrosine kinase Axl as a mediator of glioma growth and invasion. We demonstrate that Axl and its ligand Gas6 are overexpressed in human glioma cell lines and that Axl is activated under baseline conditions. Furthermore, Axl is expressed at high levels in human malignant glioma. Inhibition of Axl signaling by overexpression of a dominant-negative receptor mutant (AXL-DN) suppressed experimental gliomagenesis (growth inhibition >85%, P < 0.05) and resulted in long-term survival of mice after intracerebral glioma cell implantation when compared with Axl wild-type (AXL-WT) transfected tumor cells (survival times: AXL-WT, 10 days; AXL-DN, >72 days). A detailed analysis of the distinct hallmarks of glioma pathology, such as cell proliferation, migration, and invasion and tumor angiogenesis, revealed that inhibition of Axl signaling interfered with cell proliferation (inhibition 30% versus AXL-WT), glioma cell migration (inhibition 90% versus mock and AXL-WT, P < 0.05), and invasion (inhibition 62% and 79% versus mock and AXL-WT, respectively; P < 0.05). This study describes the identification, functional manipulation, in vitro and in vivo validation, and preclinical therapeutic inhibition of a target receptor tyrosine kinase mediating glioma growth and invasion. Our findings implicate Axl in gliomagenesis and validate it as a promising target for the development of approaches toward a therapy of these highly aggressive but, as yet, therapy-refractory, tumors.